Abstract Obesity is a major co-morbidity factor in diabetes, cancer, cardiovascular, neurological and other diseases. One group of genes recently linked to obesity in humans are genes encoding the R7 family of regulator of G protein signaling proteins (RGS6, 7, 9 and 11) and Gnb5, which encodes the G protein beta subunit G?5. G?5 and R7 proteins form obligatory dimers, so that the Gnb5 knockout causes complete degradation of the entire G?5-R7 complex. This proposal studies how a deficiency in the Gnb5 and R7 genes can lead to obesity; the ultimate goal is to discover effective treatments of disorders associated with neuroendocrine pathways regulated by these genes. The proposed research plan builds upon earlier discoveries that together constitute a strong premise for this study. Ablation of one Gnb5 allele leads to obesity and metabolic syndrome in mice; this finding was later confirmed by human genetics. Subsequent mechanistic investigations in this lab identified a novel role for the G?5-R7 complex in pancreatic beta cells where G?5-R7 strongly promoted insulin secretion. Brain is the main organ regulating body weight, and the expression level of G?5-R7 is much higher than in the pancreas or any other peripheral tissues. These considerations lead to the hypothesis that G?5-RGS7 regulates body weight via its function in the CNS, where as in the insulin-secreting beta cells, G?5-R7 can regulate hormone and/or neurotransmitter secretion by neurons. In support of this hypothesis, recent studies in the lab demonstrated that local knockout of Gnb5 in the hypothalamus using LoxP/Cre technology caused a dramatic increase of body weight and adiposity in adult mice. The proposed research will develop this discovery. Specific Aim 1 will analyze the effects of Gnb5 ablation in specific hypothalamic nuclei and types of neurons on body weight and levels of various hormones. Overexpression of Gnb5 using viral-mediated gene transfer will be performed to rescue obesity. Aim 2 will determine which hormones and signaling proteins (i.e., GPCRs) are co-expressed with Gnb5 in hypothalamic neurons. Tissue and acutely cultured neurons will be examined for secretion of specific hormones and signaling. Specific Aim 3 will study molecular events affected by G?5-R7 via its knockout (CRISPR/Cas9), knockdown and overexpression in cell lines that endogenously express M3R and G?5-R7. Experiments will focus on signaling, structure-function analysis of G?5-R7 and signal-stimulated secretion utilizing pharmacological agents and biochemical and biophysical assays. Through unraveling a novel mechanism that regulates neuronal functions, this project may have a strong impact on understanding etiology of obesity and other neuroendocrine disorders.